Cell fate decisions lead to highly reproducible patterns in metazoan development. However, these decisions hinge upon the system overcoming stochastic transcriptional events at the molecular level. The goal of the proposed study is to establish the molecular mechanisms underlying transcription in living fly embryos and to understand the dynamic and stochastic nature of these mechanisms that eventually lead to precise patterns. To gain insights into the regulatory processes that span such vastly differing temporal and spatial scales-molecular transcription events vs. formation of macroscopic patterns-we need to establish a connection between DNA regulatory elements and cell fate decisions in terms of stable transcriptional output, resolved in both time and space. Therefore we will determine a relationship between regulatory DNA elements and transcription activity using quantitative measurements to define a set of parameters characterizing transcription dynamics, such as, e.g., polymerase loading and elongation rates. Such a relationship will give us a link between sequence and fate, and will allow us to formulate quantitative models that lead to predictions that are directly testable in vivo. We propose three approaches that will contribute to the establishment of these structure- function relationships at different and complementary levels: 1) we will dissect the structure of small regulatory enhancers and measure the transcription activity and output in reporter fly constructs. 2) Using genome editing we will measure the endogenous transcription activity of enhancers and combinations of enhancers for specific genes. 3) We will scale up the approach to large numbers of genes in fixed tissue to identify classes of genes according to their dynamic transcription properties. Establishing a quantitative structure-function relationship will ultimately lead us to regulate and re-engineer the transcription programs underlying development and disease processes.